Study On Virulence Factors Of Riemerella Anatipestifer

Riemerella anatipestifer infection characterized by exudative septicaemia is primarily a disease of domestic ducks.This bacterium is also isolated from other domestic poultry,such as geese,chickens,turkeys,pigeons,wild ducks and parrots and was also reported from snail craps and domesticated pigs.Now,this bacterium is a major bacterial pathogen of ducks in all over the world.Mortality varies from 10 to 30%,but it can be as high as 95%and is influenced by predisposing viral and bacterial infections.R.anatipestifer not only accounts for major economic losses in industrialized duck production due to high mortality,reduced growth rate,poor feed conversion,increased condemnations,and high treatment costs,but also causes food safety for antibacterial.In east and south-east Asia,R.anatipestifer infection has been a problem in intensive production of meat ducks since 1982.The occurrence of different serotypes of R.anatipestifer in field cases has been reported.Unfortunately,no cross-protection has been observed among different serotypes of R.anatipestifer.Drug resistant strains of R.anatipestifer are very popular and some strains are multidrug resistant.In contrast,there has been little work on the virulence factor and pathogenesis of this organism.We have studied the epidemiological characteristic of R.anatipestifer strains which liquefacted gelatin,and it has been found that the pathogenicity of R.anatipestifer related to gelatin liquefaction.To facilitate study of the pathogenesis,gelatinase was identified and verified as a potential virulence factor of R.anatipestifer.1.Isolation of R.anatipestifer from ducks and determination of gelatin liquefactionIn the study,chocolate nutrient agar was used and 548 R.anatipestifer field strains were isolated from representing 661outbreaks of Riemerellosis in China between 2004 and 2008.537 strains had pathogenicity to 10-day-old ducks and 11 strains were avirulence.The nutrient gelatin agar plate and gelatin agar plate were used to evaluate gelatin liquefaction of R.anatipestifer and it's culture filtrate,respectively.The results showed that the virulence R.anatipestifer and its culture filtrate liquefied gelatin and avirulence strains did not.When this bacterium was cultured 10 passages in vivo or 30 passages in vitro,its activity of gelatin liquefaction did not changed obviously. Zymography showed gelatinase activity in culture filtrate of gelatin liquefaction strains and there were 6 gelatinolytic bands on a darkly stained gel.The activity of gelatinase was estimated by using densitometry and there were different among R.anatipestifer strains.2.Optimal enzyme production condition for R.anatipestifer AF strain and purification of gelatinaseA quantitative assay method of gelatinase has been established and the fermentation was studied.The results showed that the suitable condition of R. anatipestifer AF strain producing gelatinase were 2%Peptone,0.5%yeast powder, 0.5%sodium chloride,initial pH 7.4,37℃and shaking culture 24 hours.At the same time,5mmol/L Ca²⁺ promoted the producing level of gelatinase.Purification protocol of active compound was designed according to typical protein purification strategy and R.anatipestifer AF strain active compound was purified with the following program.The purification of active compound was started by concentrating the supernatantfluid in an Amicon(M_t cut-off 10,000).Second, concentrate was precipitated with ammonium sulfate at 15%saturation,and then at 70%saturation.The third step was DEAE-Sepharose anion-exchange chromatography. Fourth,phenyl-sepharose CL-4B was used in hydrophobic interaction chromatography. Finally,Superdex-75 column was used in gel permeation chromatography.The active compound obtained after steps 5 was SDS-PAGE-pure.The molecular weight was about 59kDa which was determined with SDS-PAGE.From now on,the active compound was named gelatinase.The specific activity of gelatinase was 17076.0 unit per milligram protein and it was accorded to the fifth bands of culture filtrate in zymography.3.Study on biological activity of R.anatipestifer gelatinaseIn this study,duck embryo fibroblast(DEF) and 10-day-old ducklings were used to research the biological activity of R.anatipestifer gelatinase.Cytotoxicity of gelatinase was observed using the DEF.The median lethal concentration(LC₅₀) and the median growth inhibiting(GI₅₀) were 1.45U/ml and 0.16U/ml,respectively.DEF treated with gelatinase resulted in striking morphologic changes including losses of cell membrane and cytoplasm,and nucleus being condensed. Most of the clinical strains 90%(27/30) had the capability of hydrolyzing gelatin, causing disease and altering the morphology of DEF,While the rest 10%(3/30) are on the contrary.Moreover,the damage after ducklings intrajugular injection of gelatinase was studied.The results showed gelatinase not only enhanced the invasion of R. anatipestifer,increased bacteremia,shortened stage of latency and course of disease, but also raised mortality obviously.10-days old duckling was died after intrajugular injection of 5000U/ml.Injection 2000U/ml,the duckling was fall ill,but clinical symptom did not been discovered injected 500U/ml each ducklings.Ducklings treated with gelatinase resulted in drowsiness,debility,throwing head,buccal respiration, neurological symptom,liver intumesce,bleeding point on cerebral dura mater,Cerebral edema,and so on.Though light microscope and electron microscope,the pathological changes and ultrastructural changes of main organs in the duckling which was treated with 2000U/ml by intrajugular injection were observed.After 8h,there were cardiac muscle fibers coarsened,transverse striation in nubibus,interstitial substance oedema and many vacuo-luses.Liver cell cords were irregularity and liver sinusoid shrinked. Hepatocyte was swollen and there were many vacuoluses in endochylema.Spleen resulted in endotheliocyte of central arterys swelling and ablate,acinus lienalis focal bleeding and angioleukectasia,leukomonocyte apomorphosis and necro-sis.Under electron microscope,parenchymal cells such as cadiocyte,hepatocyte,splenic lymphocyte and endothelial cell of micrangium were damaged.The structure of organelles were changed and the main manifestations were that mitochondria of cadiocyte was Swollen,cell nucleus of hepatocyte was condensed and crenulated, caryotheca of splenic lymphocyte was irregularity and caryotin was enriched at ambitus.Ducklings after intrajugular injection of gelatinase at 10 days of age were studied to determine the dynamic changes of some blood indices.The results showed that the akaryocyte and its chaplet rate of C3b decreased,the 9^th hour was the ravine and they were(1.58±0.05)×10¹²Â·L^-1 and(18.5±1.32)%,respectively.The numbers of white blood cell of peripheral blood decreased,the rate of phagocytosis and index of phagocytosis to sheep red blood cell were lowest at 9h,and they were(27.7±2.0)%and 1.053±0.01, respectively.Gelatinase decreased bactericidal index of WBC significantly when compared with controls,maximal damage was seen at 9h.Leukomonocyte and the percentage of ANAE⁺-T of peripheral blood were decreased,too.The results of serum biochemical indices showed that GOT,GPT,ALP,CHOI and GLU were all obviously enhanced,but serum total complement activity(CH₅₀) was declined.Gelatinase damaged spleen of ducklings and the worst damage was discovered at 9h.At that time,the generation activity and production IgG of splenic lymphocyte were 0.185±0.022 and 0.237±0.038,respectively.Blood brain barrier dysfunction occur in ducklings injected with gelatinase,and the damages showed at higher concentration of albumin in CSF and higher permeability in blood brain barrier,Cerebral vasogenic edema,lumens rough,junction break,basement membrane rough and break.4.Gene cloning and analysisN-terminal first ten amino acids of gelatinase protein were sequenced as NTGAAEGTHG.In NCBI,Sequence alignments indicated that the same sequence was not founded.According to the analysis of gelatinase N-terminal first ten amino acid sequence,the primer encoding sequence was designed and 197bp DNA fragment was cloned by single specific primer polymerase chain reaction(SSP-PCR) from genome DNA library of R.anatipestifer AF strain.And then,Genome walking by thermal asymmetric interlaced PCR(Tail-PCR) was used and 2150bp DNA fragment was cloned.One ORF was found out and it encoded 624 amino acids.According to bioinformation,N-terminal first twenty-four amino acids were signal peptide,and mature protein has three transmembrane domain.Unfortunately,homophylic sequence was not found in NCBI.The results showed that the gelatinase gene was a new gene probably.5.Study on physico-chemical property of R.anatipestifer gelatinaseThe activities of gelatinase were tested byâ… collagen fibers,casein,bovine serum albumin V and Azocoll.As the results showed they were not substrate of gelatinase.The optimum pH and temperature of the gelatinase were found to be 7.8 and 37℃.The activity of gelatinase was retained after storing at 4℃for 24h and at 37℃for 2h,but activity was not detected after storing at 65℃for 0.1h.Enzyme testing for inhibition and activation of the gelatinase indicated that EDTA,EGTA,Cu²⁺,Cd²⁺,Mn²⁺,Hg²⁺, PMSF,ADD and NBS inhibited enzymatic activity,2-ME and DEPC did not affected activity,but Ca²⁺,Mg²⁺,Na⁺ and Zn²⁺ activated the activity.Moreover,Ca²⁺ counteracted the inhibition of EDTA and EGTA.Animal model of R.anatipestifer infection was studied in order to showing the relationship of R.anatipestifer and gelatinase.The results showed the effects of gelatinase to ducklings were similar to R.anatipestifer infection at dysfunction, pathohistology,biochemistry,and so on.All results indicated that the gelatinase was a virulence factor and it was important to the infection of R.anatipestifer.